PEPCK-insulin gene construct and transgenic mouse

ABSTRACT

The chimeric gene is directed by a promoter or fusion of promoters which preferably are regulable and activated by the diabetic process. Preferably, it is obtained by fusion of the human insulin gene to the promoter of PEPCK (P-enolpiruvate carboxiquinasa). Said promoter (fragment -460 bp to +73 bp) is fused to the flank zone 5&#39; of the human insulin gene (-170 bp to +1). The gene of the human insulin contains two coding exons E1 and E2 and two introns A and B. It also relates to an expression vector which allows the expression of insulin in cells which are different from the β-cells of the pancreas, and to a transgenic mouse which expresses said chimeric gene.

This 371 application claims the benefit of PCT/ES94/00027, filed Mar.14, 1994.

The present invention relates firstly to a chimeric gene using the geneor cDNA (complementary DNA) of insulin driven by a promoter or fusion ofpromoters.

More specifically, it relates to the design of a chimeric gene formed bythe fusion of the promoter of P-enolpgruvate carboxyquinase to thestructural gene of human insulin, which allows the production of humaninsulin, physiologically regulated, in a tissue different from thepancreas.

The invention further relates to others objects which are describedbelow.

BACKGROUND OF THE INVENTION

Patients suffering from insulin dependent diabetes mellitus (IDDM) (typeI) depend dramatically on the administration of the hormone. Theinterruption of the insulin administration results first inhyperglycemia and ketoacidosis, then coma and finally death if thehormone is not injected. Therefore, the life and the quality of life ofthese patients depend completely on the fluctuations of the insulinlevels in their blood.

Gene therapy consists in the transfer of genetic material into cells ofa patient with the purpose of treating an illness. At present, differentapproximations of gene therapy are being developed, based on theintroduction of genes directly into animals or cells which are thentransplanted.

However, the most important goal is not to be able to transplantsuccessfully cells expressing the gene in an animal, but to make itpossible for the gene to express in a regulated and physiologic way. Thechoice of a good promoter which drives the expression of the suitablegene is crucial in order to obtain suitable plasmatic levels of thecorresponding protein.

In the case of diabetes, the question is to chose the promoter whichdrives the expression of the gene in order to obtain suitable insulinplasmatic levels for every condition of the individual. Theoverexpression of the insulin gene would result in hypoglucemia and alow expression of said gene would not modify the high glucose levels inthe diabetic process.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to better understand the disclosure herein, we enclose somedrawings concerning examples of embodiments.

In said drawings:

FIG. 1 shows the construction of the PEPCK/insulin chimeric gene fromthe plasmid pB7.0 and the human insulin gene.

FIG. 2 shows the structure of PEPCK/insulin chimeric gene.

FIG. 3 shows the process for the obtention of the vPCK/Ins retroviralvector from the pPCK/Ins plasmid.

DESCRIPTION OF THE INVENTION

One object of the invention is a chimeric gene using the gene or cDNA(complementary DNA) of insulin driven by a promoter or fusion ofpromoters, preferably adjustable and activated by the diabetic process.

Preferably, the object of the invention is a chimeric gene which isobtained by fusion of the human insulin gene to the promoter of theP-enolpiruvate carboxyquinase (PEPCK).

P-enolpyruvate carboxyquinase is a key enzyme for the control of thegluconeogenic via, and it is found mainly in the liver, kidney, jejunumand adipose tissue. The activity of this enzyme is regulated as regardsthe expression of its gene. (Hanson, R. W., et al. (1976)Gluconeogenesis: Its Regulation in mammalian Species. John Wiley & Sons,Inc., New York). The expression of the gene of PEPCK is finely regulatedby hormones. Within the fragment of the promoter of PEPCK used (-550 bpto +73 bp) sequences responding to AMPc, glucocorticoids and insulinhave been described (Wynshaw-Boris, A. et al. (1984) J. Biol. Chem. 259,12161-12169; Wynshaw-Boris, A., et al. (1986) J. Chem. 261, 9714-9720;Short, J. M., et al. (1986) J. Biol. Chem. 261, 9721-9726; O'Brien, R.M., et al. (1990) Science 249, 533-537). The glucagon, acting via AMPC,and the glucocorticoids activate the gene expression, while insulininhibits said expression. The gene expression of the PEPCK is increasedin diabetic animals due to the rise in the plasmatic levels of glucagonand the drop in the insulin levels (Tilghman, S. M., et al. (1974) Proc.Natl. Acad. Sci. USA 71, 1304-1308; Hopgood, M. F., et al (1973)Biochem. J. 134, 445-453; Kioussis, D., et al. (1978) J. Biol. Chem.253, 4327-4332). This fragment of the promoter of PEPCK is able to drivethe gene expression of the bovine growth hormone, in a regulated andspecific way, of tissue in transgenic animals (NcGrane, MlM., et al.(1988) J. Biol. Chem. 263, 11443-11451; Short, M. K., et al. (1992) Mol.Cell. Biol. 12, 1007-1020; Eisenberger, C. L. (1992) Mol. Cell. Biol.12, 1396-1403).

Therefore, when fusing to the human insulin gene this promoter of PEPCKinsulin will be produced in the tissues where the promoter of PEPCK isexpressed. In a diabetic animal the chimeric gene will be transcribed,but when sufficiently sintetized, the insulin itself will inhibit thepromoter of PEPCK which drives its expression.

So the object of the invention is the creation of a PEPCK/insulinchimeric gene. In this chimeric gene, the fragment corresponding to theinsulin gene keeps 170 bp of the flank zone 5'. The PEPCK/insulinchimeric gene contains two beginnings of transcription, onecorresponding to the insulin promoter, and the other one the promoter ofPEPCK.

It is actually a chimeric promoter, to which the portion of the promoterof PEPCK confers tissue specificity (mainly liver, kidney, jejunum andadipose). This chimeric gene has been introduced into hepatoma cells inculture and into hepatocytes in primary culture by transitorytransfection, the appearance in these cells of insulin specific mRNA inliver and immunoreactive insulin in the culture medium being observed.

Another object of the invention is a method for fusioning promoters orgene regulating elements which allows to express insulin in cell typesdifferent from the β-cells of the pancreas. In the chimeric gene, theportion of the promoter of PEPCK confers tissue specificity (mainlyliver, kidney, jejunum and adipose).

Also an object of the invention is an expression vector which allows toexpress insulin in cells different from the β-cells of the pancreas,more particularly, a pPCK/Ins plasmid vector and a vPCK/Ins retroviralvector. These vectors allow the expression of the chimeric gene incells. Other vectors of viral expression (adenovirus, herpes, virus,papillomavirus, etc.) or non-viral can be used.

They have been used to infect different types of cells in primaryculture and established cell lines (hepatoma, fibroblasts, myoblasts,preadipocytes). The infected cell lines express human insulin in apredictable way.

Further, the object of the invention is a transgenic animal expressingthe chimeric gene described above, as well as a cell type originatedfrom this transgenic animal.

Transgenic mice have been obtained in the laboratory by means of thetechnique of microinjection of the chimeric gene into fertilized mouseovules before the fusion of both the female and male pronuclei. Theanimals obtained were healthy and normoglycemic, showing that there is agood control in the regulation of the chimeric gene.

Another object of the invention is the chromosome of the transgenicanimal containing the above described chimeric gene.

Finally, the object of the invention is a chimeric gene of the abovedescribed type, for using in the gene therapy of diabetes, morespecifically, for using in the gene therapy of diabetes in tissuesdifferent from the pancreas in mammals, particularly, in the humanspecies.

Also, the invention relates to a transgenic animal of the type disclosedfor using in the development of gene therapy protocols.

EXAMPLES

For the construction of the PEPCK/insulin chimeric gene (FIG. 1) westarted from plasmid pB7.0 which contains the complete gene of PEPCK. Bymeans of digestion with the XbaI and BglII enzymes (fragment from -460bp to +73 bp), the flank zone 5' of this gene was obtained. Thisfragment was then subcloned in the polylinker of the pTZ18 plasmid. Tothis end, this plasmid was driven with XbaI and BglII and the fragmentof the promoter was later linked to these target. So the promoted ofPEPCK had been subcloned in pTZ18 and there were still some targets inthe polylinker that could later be used. Then the human insulin gene wasintroduced. In this case, the complete gene of insulin was cut withBglII and SphI and the fragment -170 bp to +1561 bp was obtained (Bell,I. B., et al (1980) Nature 284, 26-32). The pTZ18 plasmid, whichcontained the promoter of PEPCK, was driven with BglII, a targetsituated at the end of the promoter (end 3'), and SphI, a targetsituated on the polylinker. This linearized plasmid was linked to thefragment BglII/SphI of the insulin gene, and the PEPCK/insulin chimericgene was obtained, subcloned in pTZ18 (FIG. 1). This plasmid was namedpPCK/Ins.

FIG. 2 shows, in detail, the structure of the PEPCK/insulin chimericgene. The promoter of PEPCK (fragment -460 bp to +73 bp) is fusioned toflank zone 5' of the human insulin gene (-170 bp to +1). This fragmentof the promoter of insulin contains the elements recognized by thegeneral machinery of transcription and a *response element to AMPc(which induces the expression of the gene). Therefore, in the flank zone5' of the chimeric gene were two TATA box and two beginnings of thetranscription, one at the end of the promoter of PEPCK and another oneat the end of the promoter of insulin. The human insulin gene containsthree exons (two codifiers, E1 and E2) and two introns (A and B). Inposition 5' with respect to the intron A is the cap site and in 3' ofthe last exon is the polyadenylation signal (FIG. 2).

This chimeric gene has been introduced in hepatoma cells in culture andin hepatocytes in primary culture by transitory transfection of thepPCK/Ins plasmid, the appearance of insulin specific mRNA in these cellsand immunoreactive insulin in the culture medium being observed.

Once verified that the PEPCK/insulin chimeric gene was expressed in apredictable way we went over to the obtention of a retroviral vectorcontaining the chimeric gene, and which, in turn, is expressed in a wayregulated and controllable by the expression product itself, insulin.This is an essential requirement for producing a vector useful in genetherapy.

As schematically shown in FIG. 3, the fragment EcoRI-HindIII of thepPCK/Ins plasmid was subcloned for the construction of the retroviralvector, said fragment containing the complete PEPCK/insulin chimericgene, to the plasmid p12N. The Cla I fragment of the p12NPCK/Ins wasthen obtained, containing the chimeric gene, and introduced in the Cla Itarget of the pLJ(-SV40) retroviral vector (derivative from the pLJparental vector (Korman, A. J., et al. (1987) Proc. Natl. Acad. Sci. USA84, 2150-2154)). The resulting vector, vPCK/Ins, contains thePEPCK/insulin chimeric gene in an orientation opposite to the LTR 5'(promoter of the retrovirus).

This vector was introduced in psi-2 cells (Mann, R., (1983) Cell 33,153-159) by precipitation with calcium phosphate. The cells were exposedto the selection antibiotic G418 at 48 hours after transfection, andthose which were positive to the integration of the vector survived thetoxic. These cells were kept in culture for a month, and when theyreached confluence the culture medium was collected, which contained thedefective virions.

The process for obtaining a transgenic animal expressing thePEPCK/insulin chimeric gene is briefly described below.

Once verified that the chimeric gene was functional by transfection ofculture cells, the chimeric gene was then microinjected (XbaI-SphIfragmento) to mouse fertilized ovules before the fusion of both thefemale and male pronuclei according to the method described in Wagner,et al. (1981) Proc. Natl. Acad. Sci. USA 78, 5016. These embryos werethen transferred to a receptor mother. DNA of the obtained animals wasisolated from a tail fragment and then the presence of the transgene wasanalyzed by Southern blot and further hybridization with a specificprobe containing a fragment of the microinjected chimeric gene. With thetransgenic animals colonies have been established, in which theexpression of the chimeric gene has been analyzed. So the transgenicanimals obtained express the transgene in those tissues where PEPCK isussually expressed. The obtained animals were healthy and normoglycemic,thus showing that there is a good control in the regulation of thechimeric gene.

We claim:
 1. A gene construct comprising a PEPCK promoter in operablelinkage with a human insulin gene, or the complement of said geneconstruct.
 2. An expression vector comprising the gene construct ofclaim
 1. 3. The expression vector of claim 2 which is the plasmid vectorpPCK/Ins.
 4. A non-islet host cell transformed by the expression vectorof claim
 3. 5. The host cell of claim 4 that is selected from the groupconsisting of a liver cell and a hepatoma cell.
 6. The expression vectorof claim 2 which is the retroviral vector vPCK/Ins.
 7. A non-islet hostcell transformed by the expression vector of claim
 6. 8. The host cellof claim 7 that is selected from the group consisting of a liver celland a hepatoma cell.
 9. A non-islet host cell transformed by the geneconstruct of claim
 1. 10. The host cell of claim 9 that is selected fromthe group consisting of a liver cell and a hepatoma cell.
 11. The geneconstruct of claim 1, wherein the PEPCK promoter consists essentially ofa PEPCK fragment ranging from -460 bp to +73 bp as defined by FIG. 2.12. A transgenic mouse whose genome comprises a transgene comprising aPEPCK promoter operably linked to a human insulin gene, whereinexpression of said insulin gene in the tissues of said mouse isphysiologically regulated such that insulin expression by said gene isdownregulated in the presence of physiological concentrations ofextracellular insulin resulting in a state of normoglycemia in saidmouse.
 13. The transgenic mouse of claim 12, wherein insulin expressionby said gene is upregulated in the presence of glucagon resulting in astate of normoglycemia in said mouse.